U.S. patent number 10,844,411 [Application Number 15/112,886] was granted by the patent office on 2020-11-24 for inhibitors of sterol metabolism for their use to accumulate triglycerides in microalgae, and methods thereof.
This patent grant is currently assigned to Centre National De La Recherche Scientifique, Commissariat A L'Energie Atomique Et Aux Energies Alternatives. The grantee listed for this patent is Centre National de la Recherche Scientifique, Commissariat A L'energie Atomique Et Aux Energies Alternatives. Invention is credited to Caroline Barette, Jean-Christophe Cintrat, Melissa Conte, Lina-Juana Dolch, Denis Falconet, Juliette Jouhet, Eric Marechal, Coline Mei, Dimitris Petroutsos, Fabrice Rebeille.
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United States Patent |
10,844,411 |
Conte , et al. |
November 24, 2020 |
Inhibitors of sterol metabolism for their use to accumulate
triglycerides in microalgae, and methods thereof
Abstract
The invention relates to a method for accumulating
triacylglycerols in microalgae by inhibiting the sterol metabolism,
by incubating the microalgae with an inhibitor of sterol
metabolism. The invention also relates to a method for producing
fatty acids, biofuels, pharmaceutical or cosmetic compositions, and
also food supplements, comprising a triacylglycerols accumulation
step in microalgae according to the invention. Finally, the
invention concerns the use of an inhibitor of sterol metabolism to
accumulate triglycerides in microorganisms, and preferably
microalgae.
Inventors: |
Conte; Melissa (Grenoble,
FR), Dolch; Lina-Juana (Grenoble, FR), Mei;
Coline (Grenoble, FR), Barette; Caroline
(Sassenage, FR), Petroutsos; Dimitris (Grenoble,
FR), Falconet; Denis (Eybens, FR), Jouhet;
Juliette (Seyssinet, FR), Rebeille; Fabrice
(Voreppe, FR), Cintrat; Jean-Christophe (Igny,
FR), Marechal; Eric (Grenoble, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Commissariat A L'energie Atomique Et Aux Energies Alternatives
Centre National de la Recherche Scientifique |
Paris
Paris |
N/A
N/A |
FR
FR |
|
|
Assignee: |
Commissariat A L'Energie Atomique
Et Aux Energies Alternatives (Paris, FR)
Centre National De La Recherche Scientifique (Paris,
FR)
|
Family
ID: |
1000005205825 |
Appl.
No.: |
15/112,886 |
Filed: |
January 27, 2015 |
PCT
Filed: |
January 27, 2015 |
PCT No.: |
PCT/IB2015/050614 |
371(c)(1),(2),(4) Date: |
July 20, 2016 |
PCT
Pub. No.: |
WO2015/111029 |
PCT
Pub. Date: |
July 30, 2015 |
Prior Publication Data
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|
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Document
Identifier |
Publication Date |
|
US 20160348138 A1 |
Dec 1, 2016 |
|
Foreign Application Priority Data
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|
|
|
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Jan 27, 2014 [EP] |
|
|
14305111 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P
7/649 (20130101); C10L 1/02 (20130101); C12P
7/6463 (20130101); A23L 33/12 (20160801); C10L
2290/26 (20130101); C10L 2290/544 (20130101); Y02E
50/10 (20130101); A23V 2002/00 (20130101); C10L
2200/0469 (20130101) |
Current International
Class: |
C12P
7/64 (20060101); C10L 1/02 (20060101); A23L
33/12 (20160101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2012086940 |
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Aug 2012 |
|
KR |
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WO 2013/138523 |
|
Sep 2013 |
|
WO |
|
Other References
Lai et al . "Biotransformation and Bioconcentration of steroid
estrogens by Chlorella vulgaris". Applied and Environmental
Microbiology. Feb. 2002, vol. 68, No. 2, pp. 859-864. cited by
examiner .
Ying Liu et al. "Cellular responses, biodegradation and
bioaccumulation of endocrine disrupting chemicals in marine diatom
Navicula incerta". Chemosphere 2010, 80, pp. 592-599. cited by
examiner .
A.K. Franz et al., "Phenotypic Screening with Oleaginous Microalgae
Reveals Modulators of Lipid Productivity," ACS Chemical Biology,
vol. 8, 2013, pp. 1053-1062. cited by applicant .
Wei-Luen Yu et al., "Modifications of the metabolic pathways of
lipid and triacylglycerol production in microalgae," Microbial Cell
Factories, Biomed Central, London, NL, vol. 10, No. 1, Nov. 2,
2011, p. 91. cited by applicant .
Emily M. Trentacoste et al., "Metabolic engineering of lipid
catabolism increases microalgal lipid accumulation without
comprising growth," Proceedings of the National Academy of
Sciences, vol. 110, No. 49, Nov. 18, 2013, pp. 19748-19753. cited
by applicant .
Yusuf Chisti, "Biodiesel from microalgae beats bioethanol," Trends
in Biotechnology, vol. 26, No. 3, Jan. 24, 2008, pp. 126-131. cited
by applicant .
Xiaodong Deng et al., "Effect of the expression and knockdown of
citrate synthase gene on carbon flux during triacylglycerol
biosynthesis by green algae Chlamydomonas reinhardtii," BMC
Biochemistry, Biomed Central, vol. 14, No. 1, Dec. 30, 2013, p. 38.
cited by applicant .
International Search Report and Written Opinion from International
Application No. PCT/IB2015/050614, dated Apr. 8, 2015. cited by
applicant .
Belter, A. et al., Squalene Monoxygenase--A Target for
Hypercholesterolemic Therapy, Biol. Chem., vol. 392 (Dec. 2011)
1053-1075. cited by applicant .
Berman, J. D. et al., Effects of Ketoconazole on Sterol
Biosynthesis by Leishmania Mexicana, Mexicana Amastigotes in Murine
Macrophase Tumor Cells, Molecular and Biochemical Parasitology, 20
(1986) 85-92. cited by applicant .
Office Action for European Application No. 14305111.8 dated Nov.
23, 2018, 3 pages. cited by applicant.
|
Primary Examiner: Afremova; Vera
Attorney, Agent or Firm: Alston & Bird LLP
Claims
The invention claimed is:
1. A method for triggering triacylglycerols accumulation in
microalgae by inhibiting the sterol metabolism, wherein the method
comprises a step of incubating the microalgae with an inhibitor of
sterol metabolism, said inhibitor being a compound of formula (I)
or a salt thereof: ##STR00012## wherein: R.sub.41 and R.sub.42,
identical or different, represent a hydrogen atom, alkyl, alkenyl,
alkynl, or hydroxyl, --COR.sub.4a or COOR.sub.4a group, in which
R.sub.4a represents a hydrogen atom, a linear or branched alkyl,
aryl, heteroaryl group, optionally substituted with one or more
groups independently selected from alkyl or cycloalkyl groups, or
R.sub.41 and R.sub.42 form together an oxygen atom attached by a
double bond; R.sub.43 represents a hydrogen atom, or an alkyl
group; and R.sub.44, R.sub.45 and R.sub.46, identical or different,
represent a hydrogen atom, an alkyl, alkoxy, hydroxyl group, or an
oxygen atom attached by a double bond, said alkyl group being
optionally substituted with one or more halogen atoms, and
including optionally in its chain one or more sulfoxide functions,
wherein the concentration of the inhibitor of sterol metabolism
ranges from 1 .mu.M to 1M, and wherein the microalgae is selected
from the group consisting of diatom microalgae species
Phaeodactylum tricornutum and the Chromalveolata micro-algae
species Nannochloropsis gaditana.
2. A method according to claim 1, wherein the incubation step is
implemented in a nitrogen medium.
3. A method according to claim 1, wherein the inhibitor of sterol
metabolism is selected from the group consisting of
Ethinylestradiol and Estrone.
4. A method for producing fatty acids comprising a triggering of
triacylglycerols accumulation step in microalgae as defined
according to claim 1.
5. A method for producing biofuels comprising the following steps;
(i) a triggering of triacylglycerols accumulation step in
microalgae as defined according to claim 1, followed by (ii) an
extraction step of the triacylglycerols accumulated in microalgae
during step (i), and (iii) a trans-esterification step of the
triacylglycerols recovered during step (ii).
6. The method according to claim 1, wherein the concentration of
the inhibitor of sterol metabolism ranges from 5 .mu.M to 1M.
7. The method according to claim 1, wherein the concentration of
the inhibitor of sterol metabolism ranges from 5 to 20 .mu.M.
Description
FIELD
The invention relates to a method for accumulating triacylglycerols
in microalgae by inhibiting the sterol metabolism. The invention
also relates to a method for producing fatty acids, biofuels,
pharmaceutical or cosmetic compositions, and also food supplements,
comprising a triacylglycerols accumulation step in microalgae
according to the invention. Finally, the invention concerns the use
of an inhibitor of sterol metabolism to accumulate triglycerides in
microorganisms, and preferably microalgae.
BACKGROUND
It is acknowledged that oilseed production from crops cannot be
diverted from nutritional purpose (Durrett et al., The Plant
Journal (2008) 54, 593-607). Therefore, efforts are directed
towards the oil production in other organisms like algae (Chisti,
Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in
Biotechnology, 2008, Vol 26, No. 3; Dismukes et al., Current
Opinion in Biotechnology 2008, 19:235-240; Scott et al., Current
Opinion in Biotechnology 2010, 21:277-286; Singh et al.,
Bioresource Technology 102 (2011) 26-34). Studies undergone on
triacylglycerol (TAG, also called oil) production in algae (Table 1
below) have focused on the increase of TAG in cytosolic
droplets.
TABLE-US-00001 TABLE 1 Oil content of some algae (from Chisti,
Biotechnology Advances 25 (2007) 294-306) Microalga Oil content (%
dry wt) Botryococcus braunii 25-75 Chlorella sp. 28-32
Crypthecodinium cohnii 20 Cylindrotheca sp. 16-37 Dunaliella
primolecta 23 Isochrysis sp. 25-33 Monallanthus salina >20
Nannochloris sp. 20-35 Nannochloropsis sp. 31-68 Neochloris
oleoabundans 35-54 Nitzschia sp. 45-47 Phaeodactylum tricornutum
20-30 Schizochytrium sp. 50-77 Tetraselmis sueica 15-23
The advantages of microalgae over land plants have been summarized
in the EPOBIO report (Micro- and macro-algae: utility for
industrial application, September 2007, Editor: Dianna Bowles).
Both plants (crops) cultivable on arable lands and microalgae grown
in open ponds or in confined reactors are potential sources of TAG
and fatty acids for industrial purposes and biofuels (Dismukes et
al., Current Opinion in Biotechnology 2008, 19:235-240). However,
serious concerns have been raised by the intensive agricultural
practices the use of crops implies and the diversion of crops from
food to non-food chains. Efforts are thus needed to develop novel
generation biofuels based on photosynthetic microorganisms.
The main advantages of microalgae in relation to plants for the
production of TAG are the following: This bioresource does not
compete with the agro-resources used for animal or human nutrition.
Algal growth can be monitored in controlled and confined conditions
in an environmental friendly process using recycled inorganic and
organic wastes generated by other human activities and the use of
microalgae allows trapping and converting industrial byproduct
gases (e.g. CO.sub.2) into valuable organic molecules (Chisti,
Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in
Biotechnology, 2008, Vol 26, No. 3; Chisti, Journal of
Biotechnology 167 (2013) 201-214). The algal biomass productivity
is high, microalgae showing a very high potential of productivity
with cost savings when compared to land plants (see Table 2). Their
yield is variable and determined by the culturing approach
employed: it is relatively low in open pond systems while it can be
significantly increased in closed photobioreactors where culture
parameters can be controlled. This bioresource does not depend on a
geographical location, or on a season.
TABLE-US-00002 TABLE 2 Comparison of biomass productivity of major
crops ("C3" or "C4" type photosynthesis) and microalgae (extract
from the EPOBIO project report, University of York (September
2007), Table 4). "C4" crops (sorghum, "C3" crops Micro- maize,
(wheat, algae sugarcane . . . ) sunflower . . . ) Maximal
productivity (T ha.sup.-1 y.sup.-1) Microalgae (photobioreactors)
130 to 150 -- -- Higher plants -- 72 30 (maximum productivity)
Average productivity in production systems (T ha.sup.-1 y.sup.-1)
Microalgae (large scale) 10 to 50 -- -- Higher plants (field) -- 10
to 30 8 to 18 Biomass production costs 0.4-40 0.04 0.04 (USD
kg.sup.-1)
The economic viability of this sector of bioindustry is challenged
by the current limitation to combine the overall biomass yield,
i.e. dry weight of algal organic matter produced per liter and the
proportion of valuable molecules, i.e. sufficiently high proportion
of TAG per dry weight for industrial extraction and processing
(Chisti, Biotechnology Advances 25 (2007) 294-306; Chisti, Trends
in Biotechnology, 2008, Vol 26, No. 3; Chisti, Journal of
Biotechnology 167 (2013) 201-214).
In particular, the lipid composition of microalgae is compatible
with biodiesel production (Dismukes et al., Current Opinion in
Biotechnology 2008, 19:235-240; Scott et al., Current Opinion in
Biotechnology 2010, 21:277-286). The rationale for producing
biodiesel from microalgae is to use sunlight to convert water and
carbon dioxide into biomass. This biomass is then specifically
redirected towards the synthesis of oil for the generation
biofuels, by applying external stimuli like nutrient stresses,
and/or by genetic engineering of metabolism (Dorval Courchesne et
al., Journal of Biotechnology 141 (2009) 31-41).
The three most important classes of micro-algae in terms of
abundance are the diatoms (Bacillariophyceae), the green algae
(Chlorophyceae), and the golden algae (Chrysophyceae) (EPOBIO
definition). Diatoms are a major phylum of the phytoplankton
biodiversity in oceans, fresh water and various soil habitats. They
are responsible for up to 25% of the global primary productivity.
Study of this group of eukaryotes has benefited from recent
developments on Phaeodactylum tricornutum, a model of pennate
diatoms. Diatoms, like other microalgae, are considered a plausible
alternative source of hydrocarbons to replace fossil fuels or
chemicals from petrochemistry, with the advantage of having a
neutral CO.sub.2 balance, based on the hypotheses that CO.sub.2 and
water can be efficiently converted into biomass by photosynthesis
and that the carbon metabolism could be controlled so that they
accumulate energetically-rich TAG. Different phytoplanktonic
organisms of the Chromalevolata superphylum have focused the
attention for their ability to accumulate TAG, with promising
initial yields and appropriate robustness and physical properties
to be implemented in an industrial process, including Phaeodactylum
tricornutum. Phaeodaetylum tricornutum is currently used for the
industrial production of omega-3 polyunsaturated fatty acids but
industrial implementation for this application and for other
applications such as biofuels is still limited by the growth
retardation and low yield in biomass when TAG accumulation is
triggered using conventional nutrient starvation approaches, such
as nitrogen starvation (Chisti, Journal of Biotechnology 167 (2013)
201-214). These approaches have an important drawback which is the
limitation of growth that the nitrogen starvation induces.
Phaeodactylum tricornutum exhibits interesting properties for an
industrial implementation, like the ability to grow in the absence
of silicon or the sedimentation of cells that could be useful for
harvesting techniques. Attempts to promote TAG accumulation can
rely on various strategies that can be combined, including the
stimulation of fatty acid and TAG biosynthesis, the blocking of
pathways that diverts carbon to alternative metabolic routes and
eventually the arrest of TAG catabolism. Small molecules could act
on each of these three aspects of TAG metabolism.
It is possible to promote the accumulation of oil in microorganisms
by inhibiting or blocking metabolic pathways that direct the carbon
fluxes to alternative metabolites. For instance, it is well known
that blocking the accumulation of carbohydrate in storage sugars
such as starch, promotes the accumulation of oils (Siaut et al.,
BMC Biotechnology 2011, 11:7).
However, there remains a need for alternative methods to trigger
the accumulation of oil when algae are grown in a nitrogen-rich
medium, and by trying to avoid blocking carbohydrate storage
metabolism. Indeed, the carbohydrates produced after CO.sub.2
photosynthetic conversion serve as a source of carbon for all other
organic molecules within the cell, so blocking their storage has a
very strong negative impact on cell growth. Other metabolic
pathways using carbon might be blocked and allow a redirection of
carbon metabolism towards TAG metabolism.
SUMMARY
In this aim, the Inventors have now identified that the metabolism
of sterols is an alternative sink of carbon, its inhibition in
microalgae triggering the accumulation of oils.
Therefore, a first subject of the invention is a method for
triggering TAG accumulation in microalgae by inhibiting the sterol
metabolism, preferably by inhibiting the synthesis of the sterols,
said method overcoming the disadvantages listed above by incubating
the microalgae with an inhibitor of sterol metabolism.
Within the framework of the invention, the term microalgae refers
to microalgae for eukaryotes.
Also in the sense of the present invention, the TAG is built by
esterification of a 3-carbon glycerol backbone at positions 1, 2
and 3 by fatty acids. Below, TAG is synthesized by esterification
of a glycerol backbone by three fatty acids (R.sub.1, R.sub.2,
R.sub.3).
##STR00001##
In the sense of the present invention: Alkyl groups are chosen
among (C.sub.1-C.sub.26)alkyl groups, preferably
(C.sub.1-C.sub.18)alkyl groups, and more preferably
(C.sub.1-C.sub.6)alkyl groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl radicals;
Alkenyl groups are chosen among hydrocarbon chains of 2 to 26
carbon atoms, preferably 2 to 18, and more preferably 1 to 6,
having at least one carbon-carbon double bond. Examples of alkenyl
groups include ethenyl, propenyl, isopropenyl, 2,4-pentadienyl;
Alkynyl groups are chosen among hydrocarbon chains of 2 to 26
carbon atoms, preferably 2 to 18, and more preferably 1 to 6,
having at least one carbon-carbon triple bond; Alkylalkenyl means
any group derived from an alkenyl group as defined above wherein a
hydrogen atom is replaced by an alkyl group; Alkynylalkenyl means
any group derived from an alkenyl group as defined above wherein a
hydrogen atom is replaced by an alkynyl group; Cycloalkyl groups
refer to a monovalent cyclic hydrocarbon radical preferably of 3 to
7 ring carbons. The cycloalkyl group can have one or more double
bonds and can optionally be substituted. The term "cycloalkyl"
includes, for examples, cyclopropyl, cyclohexyl, cyclohexenyl and
the like; Heteroalkyl groups mean alkyl groups as defined above in
which one or more hydrogen atoms to any carbon of the alkyl is
replaced by a heteroatom selected from the group consisting of N,
O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge. The bond between the
carbon atom and the heteroatom may be a single or a double bond.
Suitable heteroalkyl groups include cyano, benzoyl, methoxy,
acetamide, borates, sulfones, sulfates, thianes, phosphates,
phosphonates, and the like; Alkoxy groups are chosen among
(C.sub.1-C.sub.20)alkoxy groups, and preferably
(C.sub.1-C.sub.4)alkoxy groups such as methyloxy, ethyloxy,
n-propyloxy, iso-propyloxy, n-butyloxy, sec-butyloxy, tert-butyloxy
and isobutyloxy radicals; Aryl groups means any functional group or
substituent derived from at least one simple aromatic ring; an
aromatic ring corresponding to any planar cyclic compound having a
delocalized .pi. system in which each atom of the ring comprises a
p-orbital, said p-orbitals overlapping themselves. More
specifically, the term aryl includes, but is not limited to,
phenyl, biphenyl, 1-naphthyl, 2-naphthyl, anthracyl, pyrenyl, and
the substituted forms thereof; Heteroaryl groups means any
functional group or substituent derived from at least one aromatic
ring as defined above and containing at least one hetero atom
selected from P, S, O and N. The term heteroaryl includes, but is
not limited to, furan, pyridine, pyrrole, thiophene, imidazole,
pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole,
pyridazole, pyridine, pyrazine, pyrimidine, pyridazine,
benzofurane, isobenzofurane, indole, isoindole, benzothiophene,
benzo[c]thiophene, benzimidazole, indazole, benzoxazole,
benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline,
quinazoline, cinnoline, purine and acridine. The aryl and
heteroaryl groups of the invention comprise preferably 1 to 12
carbon atoms, and more preferably 5 or 6 carbon atoms; Arylalkyl
means any group derived from an alkyl group as defined above
wherein a hydrogen atom is replaced by an aryl or a heteroaryl
group; Arylalkenyl means any group derived from an alkenyl group as
defined above wherein a hydrogen atom is replaced by an aryl or a
heteroaryl group; Arylalkynyl means any group derived from an
alkynyl group as defined above wherein a hydrogen atom is replaced
by an aryl or a heteroaryl group; Alkylaryl means any group derived
from an aryl group as defined above wherein a hydrogen atom is
replaced by an alkyl group.
According to the invention, halogen atoms are chosen among bromine,
chlorine, fluorine and iodine, and preferably bromine, chlorine and
fluorine.
The acid addition salts of the inhibitor of sterol metabolism
according to the invention may be for example chosen among
hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or
hydrogenophosphate, acetate, benzoate, succinate, fumarate,
maleate, lactate, citrate, tartrate, gluconate, methanesulphonate,
benzene-sulphonate and paratoluene-sulphonate.
According to a preferred embodiment, in the method of the invention
the inhibition of the sterol metabolism is realized by incubating
the microalgae with an inhibitor of sterol metabolism in a nitrogen
medium.
The concentration of the inhibitor of sterol metabolism may range
from 1 .mu.M to 1 M, and preferably from 5 to 20 .mu.M. The
incubation step lasts preferably from 24 to 72 hours.
The microalgae of the invention is advantageously selected from
microalgae of the diatom phylum, the Chromalveolata phylum, and the
Archaeplastidae phylum, and more advantageously from microalgae of
the diatom phylum and the Chromalveolata phylum. Preferably, the
microalgae is selected from the diatom micro-algae species
Phaeodactylum tricornutum and Thalassiosira pseudonana, the
Chromalveolata micro-algae species Nannochloropsis, and more
preferably Nannochloropsis gaditana, Nannochloropsis oceanica,
Nannochloropsis salina, and the Archaeplastidae micro-algae species
Chlamydomonas, Ostreococcus, Chlorella. More preferably, the
microalgae is selected from the diatom micro-algae species
Phaeodactylum tricornutum and Thalassiosira pseudonana, and the
Chromalveolata micro-algae species Nannochloropsis, and more
preferably Nannochloropsis gaditana, Nannochloropsis oceanica,
Nannochloropsis salina.
Sterol inhibitors consist of molecules that have an effect on any
protein activity in the biosynthesis of sterols, a pathway that
starts with the biosynthesis of HMG-CoA reductase, followed by the
biosynthesis of mevalonic acid, epoxysqualene and then all steroid
structures deriving from epoxysqualene (see FIG. 1). The
identification of compounds which inhibit sterol metabolism,
preferably with at least a decrease of 20% of total sterol content
per cell, can be achieved simply by colorimetric or fluorometric
dosage methods such as that commercialized by CellBioLabs (Total
Sterol Assay Kit, Colorimetric method, reference STA-384 or
Fluorometric method, reference STA-390), based on a treatment by
cholesterol oxidase/esterase, which has proved to be efficient on
detecting plant sterols (D. Kritchevsky and S. A. Tepper, Clinical
Chemistry, Vol. 25, No. 8, 1464-1465 (1979)), or also according to
methods such as those exemplified in the applications WO
2010/046385 and WO 97/03202.
According to an embodiment of the invention, the inhibitor of
sterol metabolism is a compound of formula (I) or a salt
thereof:
##STR00002## wherein: R.sub.41 and R.sub.42, identical or
different, represent a hydrogen atom, alkyl, alkenyl, alkynyl, or
hydroxyl, --COR.sub.4a or --COOR.sub.4a group, in which R.sub.4a
represents a hydrogen atom, linear or branched alkyl, aryl,
heteroaryl group, optionally substituted with one or more groups
independently selected from alkyl or cycloalkyl groups, preferably
C.sub.4-C.sub.6 cycloalkyl groups, or R.sub.41 and R.sub.42 form
together an oxygen atom attached by a double bond; R.sub.43
represents a hydrogen atom, or an alkyl group, preferably
C.sub.1-C.sub.3 alkyl group; and R.sub.44, R.sub.45 and R.sub.46,
identical or different, represent a hydrogen atom, alkyl, alkoxy,
hydroxyl group, or an oxygen atom attached by a double bond, said
alkyl group being optionally substituted with one or more halogen
atoms, and including optionally in its chain one or more sulfoxide
functions.
Advantageously, R.sub.41 and R.sub.42, identical or different,
represent a hydrogen atom, a nitrile, hydroxyl, C.sub.1-C.sub.2
alkyl or --COOR.sub.4a group in which R.sub.4a is in
C.sub.1-C.sub.7 and is optionally substituted by a C.sub.4-C.sub.6
cycloalkyl group, or R.sub.41 and R.sub.42 form together an oxygen
atom attached by a double bond.
According to another embodiment of the invention, the inhibitor of
sterol metabolism is a compound of formula (II) or a salt
thereof:
##STR00003## wherein: W.sub.3, X.sub.3, Y.sub.3 and Z.sub.3
represent carbon, sulphur, nitrogen or oxygen atom, and preferably
W.sub.3, X.sub.3, Y.sub.3 and Z.sub.3 represent carbon atoms;
n.sub.3 and n.sub.3', independently, are integer equal to 0 or 1,
and preferably n.sub.3 and n.sub.3' are equal to 1; R.sub.31 and
R.sub.32, identical or different, represent a hydrogen atom, linear
or branched alkyl, alkenyl, alkynyl, alkylalkenyl, alkynylalkenyl,
cycloalkyl, alkylaryl, arylalkyl, and preferably a benzyl group,
arylalkenyl, arylalkynyl, heteroalkyl, heteroaryl groups, or form
together a cycloalkyl group comprising 5 to 6 carbon atoms, one or
two carbon atoms of said cycloalkyl group being possibly replaced
by one or two heteroatoms, preferably nitrogen atoms, or one of
R.sub.31 or R.sub.32 form together with R.sub.39 a cycloalkyl group
comprising 5 to 6 carbon atoms, one or two carbon atoms of said
cycloalkyl group being possibly replaced by one or two heteroatoms,
preferably oxygen atoms, said R.sub.31 or R.sub.32 being optionally
substituted with one or more groups independently selected from
linear or branched alkyl, cycloalkyl such as
##STR00004## alkynyl, said alkynyl group being preferably a
C.sub.5-C.sub.12 branched alkynyl group, arylalkyl, aryl,
heteroaryl, hydroxyl, halogen, nitro, --COR.sub.3a or
--NR.sub.3aR.sub.3b group, in which R.sub.3a and R.sub.3b,
identical or different, represent a hydrogen atom or a linear or
branched alkyl chain; R.sub.33 represents a hydrogen atom, a linear
or branched alkyl chain such as a C.sub.1-C.sub.3 alkyl group, or a
nitrile group, and preferably R.sub.33 represents a hydrogen atom;
R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38 and R.sub.39,
identical or different, represent hydrogen or halogen atoms, or
hydroxyl groups, and preferably R.sub.34, R.sub.35, R.sub.36,
R.sub.37, R.sub.38 and R.sub.39 are hydrogen atoms.
The dotted lines of formula (II) represent single or double bonds.
Specifically, said W.sub.3--X.sub.3 bond or Y.sub.3--Z.sub.3 bond
is single bond respectively when one of W.sub.3 or X.sub.3 of the
W.sub.3--X.sub.3 bond, or one of Y.sub.3 or Z.sub.3 of the
Y.sub.3--Z.sub.3 bond, is sulphur or oxygen. Said W.sub.3--X.sub.3
bond or Y.sub.3--Z.sub.3 bond is double bond respectively when
W.sub.3 and X.sub.3, or Y.sub.3 and Z.sub.3 are carbon or
nitrogen.
According to a preferred embodiment, R.sub.31 and R.sub.32,
identical or different, represent a hydrogen atom, linear or
branched alkyl, alkenyl, alkynyl, alkynylalkenyl, cycloalkyl,
alkylaryl, arylalkyl, and preferably a benzyl group, arylalkenyl,
arylalkynyl, heteroalkyl, heteroaryl groups, or form together a
cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon
atoms of said cycloalkyl group being possibly replaced by one or
two heteroatoms, preferably nitrogen atoms, or one of R.sub.31 or
R.sub.32 form together with R.sub.39 a cycloalkyl group comprising
5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl
group being possibly replaced by one or two heteroatoms, preferably
oxygen atoms, said R.sub.31 or R.sub.32 being optionally
substituted with one or more groups independently selected from
linear or branched alkyl, cycloalkyl such as
##STR00005## alkynyl, said alkynyl group being preferably a
C.sub.5-C.sub.12 branched alkynyl group, arylalkyl, aryl,
heteroaryl, hydroxyl, halogen, nitro, --COR.sub.3a or
--NR.sub.3aR.sub.3b group, in which R.sub.3a and R.sub.3b,
identical or different, represent a hydrogen atom or a linear or
branched alkyl chain.
According to a more preferred embodiment, R.sub.31 and R.sub.32,
identical or different, represent C.sub.1-C.sub.6 linear or
branched alkyl chains, or an arylalkyl group, optionally
substituted with one or more groups independently selected from
linear or branched alkyl. Advantageously, R.sub.31 represents a
C.sub.1-C.sub.6 linear or branched alkyl chain, and R.sub.32 a
benzyl group substituted with a C.sub.1-C.sub.6 linear or branched
alkyl chain, or an alkenyl group substituted with a linear or
branched alkynyl group, such as a
--(CH.sub.2)n.sub.3''-CH.dbd.CH--C.ident.C--C(CH.sub.3).sub.3 group
in which n.sub.3'' ranges from 1 to 6.
According to another embodiment of the invention, the inhibitor of
sterol metabolism is a compound of formula (III) or a salt
thereof:
##STR00006## wherein: n.sub.1 ranges from 1 to 12, and preferably
n.sub.1=2, R.sub.11 represents a hydrogen atom; a --COR.sub.1a
group, in which R.sub.1a is a group selected from a linear or
branched alkyl chain, optionally substituted with one or more
groups independently selected from hydroxyl, halogen, nitro,
optionally substituted benzyl groups, or optionally substituted
aryl groups such as phenyl groups, said aryl groups being
eventually substituted with one or more groups independently
selected from halogen atoms and methyl groups; R.sub.12, R.sub.13
and R.sub.14, identical or different, represent a hydrogen atom; a
linear or branched alkyl chain; a hydroxyl group; a --CH.sub.2OH
group; a --COOR.sub.1b group, in which R.sub.1b represents a
hydrogen atom, or a linear or branched alkyl chain; a
--OSiR.sub.1cR.sub.1dR.sub.1e group, in which R.sub.1c, R.sub.1d
and R.sub.1e, identical or different, represent a hydrogen atom, or
a linear or branched alkyl chain; or R.sub.12 and R.sub.13 are
fused together to form an exo methylene group; R.sub.15 represents
a hydroxyl group; a --OSiR.sub.1cR.sub.1dR.sub.1e group as defined
above; a --COOR.sub.1f group, in which R.sub.1f represents a
hydrogen atom, or a linear or branched alkyl chain; a --OCOR.sub.1g
group, in which R.sub.1g represents a linear or branched alkyl
chain, preferably a C.sub.1-C.sub.3 alkyl chain, and more
preferably a C.sub.1 alkyl group; or R.sub.15 is a carbon forming
an ethylenic unsaturation with the tetrahydropyranone ring.
According to a preferred embodiment, R.sub.11 is a --COR.sub.1a
group, in which R.sub.1a is a linear or branched alkyl chain, and
preferably R.sub.11 is a --COCH(CH.sub.3)C.sub.2H.sub.5 or
--COC(CH.sub.3).sub.2C.sub.2H.sub.5 group.
Advantageously, R.sub.14 represent a C.sub.1-C.sub.6 alkyl chain,
preferably a --CH.sub.3 group, and both R.sub.12 and R.sub.13
represent hydrogen atoms. Alternatively, R.sub.12 and R.sub.14 may
represent a C.sub.1-C.sub.6 alkyl chain, preferably a --CH.sub.3
group, and R.sub.13 a hydrogen atom.
According to another preferred embodiment, n.sub.1=2, and R.sub.15
represents a hydroxyl group.
According a preferred embodiment of the invention, the inhibitor of
sterol metabolism is selected from Ethinylestradiol, Mevastatin,
Simvastatin, Butenafine, Terbinafine, Estrone. The most preferred
inhibitors of sterol metabolism of formula (I) are Ethinylestradiol
and Estrone. The most preferred inhibitor of sterol metabolism of
formula (II) is Butenafine. The most preferred inhibitors of sterol
metabolism of formula (III) are Mevastatin and Simvastatin.
The inhibitors of sterol metabolism of the invention are all acting
on the "mevalonate" pathway, and not on the "non-mevalonate"
pathway of synthesis of sterols (see FIG. 1).
More specifically: the inhibitors of sterol metabolism of formula
(I) are acting as steroid structural analogs interacting with
protein involved in sterol metabolism including Estrone (Merola and
Arnold, Science, Vol. 144, 301-302 (1964)) and Ethinylestradiol
(Koopen et al., Journal of Lipid Research, Vol. 40, 1999), the
inhibitors of sterol metabolism of formula (II) are acting as
squalene epoxidase inhibitors (Belter et al., Biol. Chem., Vol.
392, 1053-1075 (2011)), and the inhibitors of sterol metabolism of
formula (III) are acting as HMG-CoA reductase inhibitors (Liu et
al., Mol. Biol. Rep. (2010) 37:1391-1395).
Another subject-matter of the invention is a method for producing
fatty acids comprising a triggering of triacylglycerols
accumulation step in microalgae as defined according to the
invention, followed by an extraction step of the triacylglycerols
accumulated in the microalgae.
The invention also relates to a method for producing biofuels
comprising the following steps:
a triggering of triacylglycerols accumulation step in microalgae as
defined according to the invention, followed by
(ii) an extraction step of the triacylglycerols accumulated in
microalgae during step (i), and
(iii) a trans-esterification step of the triacylglycerols recovered
during step (ii), for example as described by Zhang et al.,
Bioresource Technology 147 (2013) 59-64.
The invention also concerns a method for producing pharmaceutical
or cosmetic compositions comprising the following steps:
(i') a triggering of triacylglycerols accumulation step in
microalgae as defined according to the invention, followed by
(ii') an extraction step of the triacylgycerols accumulated in
microalgae during step (i'), and
(iii') a step of adding at least one pharmaceutically or
cosmetically acceptable excipient to the triacylglycerols recovered
during step (ii').
The invention also relates to a method for producing human food and
animal feed supplements comprising the following steps:
(i'') a triggering of triacylglycerols accumulation step in
microalgae as defined according to the invention, followed by
(ii'') an extraction step of the triacylglycerols accumulated in
microalgae during step (i''), and
(iii'') a step of adding at least one food additive to the
triacylglycerols recovered during step (ii'').
The methods of the invention may comprise one or more extraction
steps after the triggering of triacylglycerols accumulation step in
microalgae. The extraction step may be implemented using solvents
or another extraction method well known form the skilled
artisan.
Another subject-matter of the invention relates to the use of an
inhibitor of sterol metabolism to accumulate triglycerides in
microorganism, preferably in microalgae, more preferably in
microalgae of the diatom phylum, and still more preferably in the
diatom microalgae species Phaeodactylum tricornutum.
Advantageously, the invention concerns the use of an inhibitor of
sterol metabolism selected from the compounds of formula (I), (II)
and (III), such as Ethinylestradiol, Mevastatin, Simvastatin,
Butenafine, Terbinafine, Estrone, to accumulate triglycerides in
microorganism, preferably in microalgae, more preferably in
microalgae of the diatom phylum, and still more preferably in the
diatom microalgae species Phaeodactylum tricornutum. The invention
also concerns the use of combination of inhibitors of sterol
metabolism selected from the compounds of formula (I), (II) and
(III), such as Ethinylestradiol, Mevastatin, Simvastatin,
Butenafine, Terbinafine, Estrone, as well as their combination with
other methods known to enhance the accumulation of TAG in
microalgae, in particular a shortage of nutrient, and more
preferably a shortage of nitrogen.
BRIEF DESCRIPTION OF THE DRAWINGS
In addition to the above provisions, the invention also comprises
other provisions which will emerge from the remainder of the
description which follows, and also to the appended drawings in
which:
FIG. 1 gives a schematic view of the sterol biosynthetic pathway
via mevalonic acid, and represents the action of Mevastatin and
Simvastatin (Liu et al., Mol. Biol. Rep. (2010) 37:1391-1395),
Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964),
Butenafine and Terbinafine (Belter et al., Biol. Chem., Vol. 392,
1053-1075 (2011)),
FIG. 2 represents pictures of a Phaeodactylum cell visualized by
confocal microscopy after a 72 h cultivation in Nitrogen-rich N(+)
or in Nitrogen-starved N(-) medium On the left side (A and C),
cells are shown in phase contrast ("phase"). On the right side (B
and D), cells are shown after excitation of Nile red at 488 nm and
emission at 519 nm,
FIG. 3a-h illustrates the dose response for Ethinylestradiol,
Mevastatin, Simvastatin and Estrone, and
FIG. 4 illustrates the dose response for Ethinylestradiol,
Mevastatin and Simvastatin with an evaluation of the TAG content by
staining with Nile Red and an evaluation of cells by counting in an
aliquote fraction using a Malassez cell,
FIG. 5 shows the effect of Butenafine on Nile Red accumulation in
Nannochloropsis gaditana grown respectively in ESAW 1N1P media and
ESAW 10N10P media. Nile Red Fluorescence was normalized by
calculating the relative fluorescence units per million cells,
and
FIG. 6 shows the effect of Ethinylestradiol on Nile Red
accumulation in Nannochloropsis gaditana grown respectively in ESAW
1N1P media and ESAW 10N10P media. Nile Red Fluorescence was
normalized by calculating the relative fluorescence units per
million cells.
DETAILED DESCRIPTION
Examples
1) Materials & Methods
1) 1-Phaeodactylum tricornutum Strain and Growth Conditions
Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (Culture
Collection of Marine Phytoplankton, now known as NCMA: National
Center for Marine Algae and Microbiota) was used in
experiments.
Pt1 was grown at 20.degree. C. in 250 mL flask using "enriched
artificial seawater" (ESAW) medium, prepared following the
recommendations of the Canadian Center for the Culture of
Microorganisms.
To prepare the ESAW medium, four separated solutions are prepared,
two solutions of salts (solutions 1 and 2), one solution of
nutrients, and one solution of vitamins. Salts are added in order
to distilled deionized water (DDW). When the salts in solutions 1
and 2 are completely dissolved, the solutions 1 and 2 are mixed
together. The total volume is diluted with DDW.
TABLE-US-00003 TABLE 3 Compositions of the ESAW salts solutions 1
and 2 Molecular weight Amount to weight Concentration (g
mol.sup.-1) (g/L solution) (mM) Solution 1: Anhydrous salts NaCl
58.44 20.756 362.7 Na.sub.2SO.sub.4 142.04 3.477 25.0 KCl 74.56
0.587 8.03 NaHCO.sub.3 84 0.17 2.067 KBr 119.01 0.0845 0.725
H.sub.3BO.sub.3 61.83 0.022 0.372 NaF 41.99 0.0027 0.0657 Solution
2: Hydrated salts MgCl.sub.2.cndot.6H.sub.2O 203.33 9.395 47.18
CaCl.sub.2.cndot.2H.sub.2O 147.03 1.316 9.134
SrCl.sub.2.cndot.6H.sub.2O 266.64 0.0214 0.082
TABLE-US-00004 TABLE 4 Nutrient Enrichment Stocks Stock
concentration Final concentration Solutions (g L.sup.-1) (.mu.M) 1
NaNO.sub.3 46.67 549.1 2* Na.sub.2 glycerophosphate 6.67 21.8 3
Na.sub.2SiO.sub.3.cndot.9H.sub.2O 15.00 105.6 4**
Na.sub.2EDTA.cndot.2H.sub.2O 3.64 9.81
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O*** 2.34 5.97
FeCl.sub.3.cndot.6H.sub.2O 0.16 0.592 5 MnSO.sub.4.cndot.4H.sub.2O
0.54 2.42 ZnSO.sub.4.cndot.7H.sub.2O 0.073 0.254
CoSO.sub.4.cndot.7H.sub.2O 0.016 0.0569
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 0.126 0.520
Na.sub.2EDTA.cndot.2H.sub.2O 1.89 5.05 6 H.sub.3BO.sub.3 3.80 61.46
7 NaSeO.sub.3 0.00173 0.001 *Na.sub.2 glycerophosphate can be
replaced with an equimolar stock of Na.sub.2HPO.sub.4.
**Na.sub.2EDTA.cndot.2H.sub.2O is added before the trace metals
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O and
FeCl.sub.3.cndot.6H.sub.2O.
***Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O can be
replaced with an equimolar stock of FeCl.sub.3. Solution 5 is
adjusted to pH = 6 with 2 g of Na.sub.2CO.sub.3. Solution 4 can be
heated to dissolve the iron.
TABLE-US-00005 TABLE 5 Vitamin Stocks Stock concentration Final
concentration Vitamin Stock (g L.sup.-1) (mM) Thiamine 0.1 2.97
.times. 10.sup.-1 Vitamin B12 0.002 1.47 .times. 10.sup.-3 Biotin
0.001 4.09 .times. 10.sup.-3
To prepare the ESAW medium, the solutions are filtered through 0.45
.mu.m membrane filter with a glass fiber prefilter. A flask is
acid-washed in 10% HCl and rinsed in distilled water before first
use. To 1 L of filtered salt solution, 1 mL of Nutrient Enrichment
Stock solutions 1, 2, 4, 5, 6 and 7, 2 mL of Nutrient Enrichment
Stock solution 3, and 2 mL of the Vitamin Stock are added (Tables 4
and 5). To reduce precipitation during autoclaving, 1.44 mL of 1N
HCl and 0.12 g of sodium bicarbonate are added. The obtained ESAW
medium is then sterilized by autoclaving.
Cells were grown on a 12:12 light (450 .mu.Einstein-1
sec.sup.-1)/dark cycle (an Einstein defined the energy in one mole
(6.022.times.10.sup.23) of photons). Cells were sub-cultured every
week by inoculate fresh media with 1/5 of previous culture.
Nitrogen-rich N(+) medium contained no source of nitrogen.
Nitrogen-starved N(-), medium contained 0.05 g/L NaNO.sub.3. To
monitor cell growth, a genetically modified strain containing a
Histone H4 protein fused to the yellow fluorescent protein was used
(Siaut et al., Gene 406 (2007) 23-35).
1) 2-Principle of Nile Red Staining of Oil Droplets
Accumulation of oil droplets can be monitored by Nile Red (Sigma
Aldrich) fluorescent staining (Excitation wavelength at 485 nm;
emission at 525 nm) as described by Ren et al. (Biotechnology for
Biofuels 2013, 6:143), Cells were diluted and adjusted to a cell
density that was linearly correlated with Nile Red fluorescence.
Nile Red solution (40 .mu.L of 2.5 .mu.gmL.sup.-1 stock
concentration, in 100% DMSO) was added to 160 .mu.L cell
suspension. Specific fluorescence was determined by dividing Nile
Red fluorescence intensity by the number of cells. Oil bodies
stained with Nile Red were then visualized using a Zeiss
AxioScope.A1 microscope (FITC filter; Excitation wavelength at 488
nm; emission at 519 nm).
Lipid droplets can be visualized. In FIG. 2, a Phaeodactylum cell
was visualized by confocal microscopy after a 72 h cultivation in
Nitrogen-rich N(+) medium or in Nitrogen-starved N(-) medium. On
the left side, cells are shown in phase contrast ("phase"). On the
right side, cells are shown after excitation of Nile red at 488 nm
and emission at 519 nm. Lipid droplets can be clearly
visualized.
This principle was use to measure the presence of oil in
Phaeodactylum tricornutum simply by using a spectrofluorometer (in
these conditions, to lower the detection of other fluorophores
within the cell, such as chlorophylls, excitation was at 530 nm and
emission at 580 nm).
1) 3-Alternative Method for Oil Level Detection
Alternatively, oil is extracted using solvents or another
extraction method, separated and purified by thin layer
chromatography and methanolyzed to produce fatty acid methyl esters
and quantified by gas chromatography coupled to a ionization flame
detector or a mass spectrometer.
TAG were extracted from 200 mg of freeze-dried Phaeodactylum
tricornutum cells in order to prevent lipid degradation. Briefly,
cells were frozen in liquid nitrogen immediately after harvest. The
freeze-dried cell pellet was resuspended in 4 mL of boiling ethanol
for 5 minutes followed by the addition of 2 mL of methanol and 8 mL
of chloroform at room temperature. The mixture was then saturated
with argon and stirred for 1 h at room temperature. After
filtration through glass wool, cell remains were rinsed with 3 mL
of chloroform/methanol (2:1, v/v). In order to initiate biphase
formation, 5 mL of NaCl 1% was then added to the filtrate. The
chloroform phase was dried under argon before re-solubilization of
the lipid extract in pure chloroform. To isolate TAG, lipids were
run on silica gel thin layer chromatography (TLC) plates (Merck)
with hexane/diethylether/acetic acid (70:30:1, v/v). Lipids were
then visualized under UV light after pulverization of
8-anilino-1-naphthalenesulfonic acid at 2% in methanol. They were
then scraped off from the TLC plates for further analyses. For acyl
profiling and quantification of TAG, fatty acids were methylated
using 3 mL of 2.5% H.sub.2SO.sub.4 in methanol during 1 h at
100.degree. C. (including standard amounts of 21:0). The reaction
was stopped by the addition of 3 mL of water and 3 mL of hexane.
The hexane phase was analyzed by gas liquid chromatography (Perkin
Elmer) on a BPX70 (SGE) column. Methylated fatty acids were
identified by comparison of their retention times with those of
standards and quantified by surface peak method using 21:0 for
calibration. Extraction and quantification were done at least 3
times.
1) 4-Principle of Cell Normalization
The number of cells in a sample can be evaluated using a
fluorescent reporter, like the strain by Siaut et al. (Gene 406
(2007) 23-35) containing a genetic construction with Histone H4
protein fused to the Yellow Fluorescent Protein (YFP) (FIG. 5A of
Siaut et al., Gene 406 (2007) 23-35).
For cell counting, we can either use this strain called "ptYFP" and
measure the fluorescence emitted by the YFP at 530 nm after
excitation at 515 nm, or use any strain and estimate cell numbers
by counting with a Malassez grid (supplier: Mareinfeld).
1) 5-Incubation of Phaeodactylum tricornutum with Inhibitors of the
Sterol Metabolism and Detection of Oil Accumulation Triggered by
the Treatment
On the first day, we prepared 48 well plates by adding 4 mm glass
beads sterilized with Ultra-Violet exposure in each well.
We prepared fresh suspensions of microalgae in exponential growth
phase. For cell normalization based on YFP fluorescence, cells of
Phaeodactylum tricornutum containing a YFP reporter (ptYFP)
cultured in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min.
For cell normalization based on counting using a Malassez grid,
cells of the Pt1 strain of Phaeodactylum tricornutum cultured in
N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. The
supernatant was discarded and the pellet suspended in N(-) ESAW.
The microalgae were then centrifuged at 3,500 rpm, 5 min. The
supernatant was discarded and the pellet suspended in N(-) ESAW.
Cells were then diluted to 1.times.10.sup.6 cells/mL in N(-) ESAW.
Samples were then separated into two batches. One was supplemented
with 1 .mu.L/mL of 46.7 g/L NaNO.sub.3 stock to obtain a suspension
of cells in N(+) ESAW medium. Another was left without NaNO.sub.3
to obtain a N(-) ESAW culture as a control for high lipid
accumulation. In a 48-well clear NUNC plate, 450 .mu.L/well of
1.times.10.sup.6 cells/mL were dispensed.
For a dose-response analysis, each well of the 48-well plate was
then subjected to an appropriate incubation with 0, 1, 10 or 100
.mu.M of inhibitor of sterol metabolism using 50 .mu.L of the
following: 50 .mu.L of a 10 time concentrated solution of inhibitor
of sterol metabolism (5% DMSO:95% N(+) ESAW) or 50 .mu.L of
Nitrogen-rich medium without any inhibitor of sterol metabolism (5%
DMSO:95% N(+) ESAW) or Nitrogen-starved medium without any
inhibitor of sterol metabolism (5% DMSO:95% N(-) ESAW) as a
positive control.
For an analysis of the effect of a single dose, an incubation was
performed with 0, and a chosen concentration of inhibitor of sterol
metabolism (50 .mu.M).
In all cases, the edge of the plate was sealed using a parafilm.
Plates were incubated for 48 h in an incubator with top lighting,
20.degree. C., 100 rpm, 12 h/12 h light/dark.
After an incubation of 48 hours, fluorescence was measured at the
following excitation/emission wavelengths, 530/580 nm (to evaluate
a baseline fluorescence prior Nile Red addition) and 515/530 nm (to
evaluate YFP fluorescence). Following this first measure, 40 .mu.L
of Nile Red (2.5 .mu.g/mL stock concentration, in 100% DMSO) are
added. Plates are mixed and incubated 20 minutes at room
temperature, protected from light. Nile Red fluorescence is then
measured using a spectrofluorometer (excitation 530 nm/emission 580
nm).
1) 6-Incubation of Nannochloropsis gaditana with Inhibitors of the
Sterol Metabolism and Detection of Oil Accumulation Triggered by
the Treatment
Experiments were performed in two different conditions of
Nannochloropsis gaditana: cells were either cultured in ESAW
containing 47 mgL.sup.-1 NaNO.sub.3 and 3 mgL.sup.-1
NaH.sub.2PO.sub.4 (medium 1N1P), or 470 mg/L NaNO.sub.3 and 30
mgL.sup.-1NaH.sub.2PO.sub.4) (medium 10N10P). Cells in an
exponential growth phase were collected via centrifugation at 3500
rpm for 10 minutes. The supernatant was discarded and the cells
were resuspended in the same volume of ESAW medium (either 1N1P or
10N10P). The cultures were centrifuged again at 3500 rpm, for 10
minutes, and the supernatant was discarded. The pellet was
resuspended in ESAW (either 1N1P or 10N10P) to obtain a
concentration of 2.times.10.sup.6 cells/mL. Cell counts were
performed using a Malassez counting chamber, allowing 10 minutes
for the cells to settle before counting.
Twenty milliliters of 2.times.10.sup.6 cells/mL of Nannochloropsis
gaditana in ESAW (10N10P) and ESAW (1N1P) were dispensed into
sterile glass conical flasks. Stocks of inhibitor of sterol
metabolism were prepared in DMSO. Inhibitors of sterol metabolism
were added to the 20 mL Nannochloropsis gaditana samples at final
concentrations of 10 .mu.M, 30 .mu.M, or 100 .mu.M. The maximum
final concentration of DMSO in the samples was 1% (v/v), All
cultures were incubated for seven days at 100 rpm, 12 h/12 h
light/dark cycle, 50 .mu.Em.sup.-2s.sup.-1, 20.degree. C.
Each day, an aliquot was taken from each flask in order to perform
a Nile Red stain and cell counts. 160 .mu.L per sample was added to
black 96 well plates, and allowed to settle for 10 minutes. In
order to detect any background noise, fluorescence was measured at
excitation and emission of 530 nm and 580 nm, respectively. 40
.mu.L of 2.5 .mu.gmL.sup.-1 Nile Red in DMSO was added to each
well, and mixed thoroughly. After 20 minutes of incubation, Nile
Red fluorescence was measured at excitation and emission of 530 nm
and 580 nm, respectively.
Nile Red Fluorescence was normalized by calculating the relative
fluorescence units per million cells. Results were expressed as a
percentage of Nile Red fluorescence of Nannochloropsis gaditana
cultured in complete medium (either ESAW (10N10P) or ESAW
(1N1P)).
1) 7-Inhibitors of Sterol Metabolism
Inhibitors of sterol metabolism were obtained from the Prestwick
library for their ability to trigger the accumulation of lipid
droplets within the cells of Phaeodactylum tricornutum, and then
purchased from Sigma-Aldrich,
TABLE-US-00006 TABLE 6 Inhibitors of sterol metabolism selected
from the Prestwick library Chemical name Structure Molecular
Formula Mevastatin ##STR00007## C.sub.23H.sub.34O.sub.5 Butenafine
##STR00008## C.sub.23H.sub.27N Simvastatin ##STR00009##
C.sub.25H.sub.38O.sub.5 Estrone ##STR00010##
C.sub.18H.sub.22O.sub.2 Ethinylestradiol ##STR00011##
C.sub.20H.sub.24O.sub.2
2) Results
Phaeodactylum tricornutum was incubated for 48 h in presence of 10
.mu.M of Mevastatin, Butenafine, Simvastatin, Estrone,
Ethinylestradiol and Terbinafine.
In all cases the presence of oil per cell increased by a factor of
at least 1.5, based on Nile Red staining.
FIG. 3a-h illustrates the dose response for Ethinylestradiol,
Mevastatin, Simvastatin and Estrone with an evaluation of the TAG
content by staining with Nile Red, and an evaluation of cells by
monitoring YFP fluorescence from an expressed Histone H4 reporter
gene. On the left, FIG. 3a-h shows the Nile Red levels in percent
of untreated cells and the number of cells estimated by the YFP
fluorescence expressed in percent of untreated cells. The right
panels show the increase of oil content per cell, by the ratio of
Nile Red fluorescence/YFP fluorescence, expressed in percent of
untreated cells. The oil content per cell increases with drug
concentration and consequently level of sterol metabolism
inhibition and ranges from 120 to 400% when compared to untreated
cells.
FIG. 4 illustrates the dose response for Ethinylestradiol (A),
Mevastatin (B) and Simvastatin (C) with an evaluation of the TAG
content by staining with Nile Red and an evaluation of cells by
counting in an aliquote fraction using a Malassez cell. In
histograms of FIG. 4, the white bars indicate the evaluation of
cell numbers in percent of untreated cells, and the black bars
indicate the Nile Red per 10.sup.6 cell, in percent of untreated
cells. The oil content per cell, and consequently level of sterol
metabolism inhibition, increases with incubation of 50 .mu.M of
inhibitor, and ranges from 120 to 350% when compared to untreated
cells.
Nannochloropsis gaditana was incubated in presence of Butenafine
and
Ethinylestradiol.
FIGS. 5 and 6 illustrate the effect of Butenafine and
Ethinylestradiol on Nile Red accumulation in Nannochloropsis
gaditana grown respectively in ESAW 1N1P media and ESAW 10N10P
media. Nile Red Fluorescence was normalized by calculating the
relative fluorescence units per million cells.
Both Butenafine and Ethinylestradiol trigger the accumulation of
oil in Nannochloropsis gaditana, in different media and with a time
course that can be observed at least for 7 days.
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